Biofuels are current favorites to be the next generation transportation fuels. They are produced from renewable biological sources such as vegetable oils and animal fats. They are biodegradable, non-toxic and have a low emission profile. Due to the limited sources of biodiesel raw materials such as rape seed oil, soy bean oil or palm oil, it is of importance to expand biodiesel raw materials to non-food materials like microbes. The benefits of using microbes for production of oils are: they are affected neither by seasons nor by climates, they are able to produce high lipid contents, and the oils can be produced from a wide variety of sources with short production times, especially from residues with abundant nutrition. Microbiologically produced lipids may also be used e.g. for the production of functional fatty acids.
A few fungal species accumulate remarkable amounts of lipid in the cells. It has been observed that lipids accumulate in these so called oleaginous fungi under nitrogen limited conditions, which has resulted in a hypothesis for effective lipid accumulation (Review Ratledge and Wynn 2002 and references thenceforth). Nitrogen limitation causes activation of the AMP deaminase which utilizes AMP to produce NH4. The decrease in AMP concentration inhibits the activity of mitochondrial isocitrate dehydrogenase (IDH) which is part of the mitochondrial tricarboxylic (TCA) cycle. Reduction in IDH activity results in equilibration of isocitrate to citrate by aconitase. Produced citrate is transferred to the cytosol where it is converted with Coenzyme A (CoA) to acetyl-CoA by ATP:citrate lyase (ACL) with ATP hydrolysis.
Cytosolic acetyl-CoA can be further used in fatty acid synthesis. A comprehensive review on fatty acid synthesis and elongation in yeast, especially in Saccharomyces cerevisiae, is that of Tehlivets et al., 1997. In the first step of fatty acid synthesis acetyl-CoA is carboxylated by the addition of carbon dioxide to malonyl-CoA by the enzyme acetyl-CoA carboxylase in an ATP demanding reaction. In the following reactions by the fatty acid synthase systems acyl and malonyl moieties from acyl-CoA and malonyl-CoA, respectively, are transferred to acyl carrier proteins (ACPs), after the acyl chain, typically initiated by acetyl-CoA, is condensated with malonyl-ACP followed by reduction of the 3-ketoacyl-ACP to 3-hydroxyacyl-ACP, dehydration to enoyl-ACP, and a second reduction to a saturated acyl-chain that is extended by two carbon atoms. These synthesis steps are usually repeated seven times resulting in palmitoyl ACP (C16:0). Palmitic acid and intermediates of the fatty acid synthesis after hydrolysed to acyl-CoAs by hydrolase/thioesterase, can be further modified by different elongases and desaturases to different length acyl-chains with or without double bonds. In one cycle of fatty acid synthesis two NADPHs are required in the reduction steps. Acyl-CoAs can be further synthesised to triacylglycerols.
Triacylglycerol synthesis starts from glycerol-3-phosphate or dihydroxyacetone phosphate which is acylated (dihydroxyacetone-phosphate also reduced) to 1-acyl-glycerol-3-phosphate which is further acylated to phosphatidic acid. Phosphatidic acid can be further dephosphorylated to diacylglycerol. Diacylglycerol is further acylated to triacylglycerol mainly by acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) utilizing acyl-CoA or phosphatidylcholine, respectively, as acyl donors. The triacylglycerol pathway in yeast S. cerevisiae is described in more detail in a mini-review of Sorger and Daum 2003.
Phospholipid:diacylglycerol acyltransferase (PDAT) encoding genes originating from S. cerevisiae and Yarrowia lipolytica have been expressed in yeasts S. cerevisiae and Y. lipolytica to enhance their triacylglycerol production (WO00/60095 and WO2005/003322, respectively). WO2009/126890 provides systems for producing engineered oleaginous yeast or fungi that express carotenoids. Oleaginy is promoted e.g. by increased or heterologous expression of DGAT or PDAT, whereas reducing the activity of PDC is expected to promote oleaginy.
Methods of manufacturing biodiesel and other oil-based compounds using glycerol as an energy source in fermentation of oil-bearing microorganisms have been described e.g. in US2009/0004715. Methods of producing lipid-based biofuels from cellulose containing feedstock by heterotrophic fermentation of microorganisms have been described in US2009/0064567. Both publications focus on the use of algae as lipid producers. No details are given. WO2007/136762 provides genetically engineered microorganisms that produce desired products from the fatty acid biosynthetic pathway.
With the above-described triacylglycerol production pathway high triglyceride yields indicated as triglyceride production per used carbon source cannot be achieved or triacylglycerol production per cell biomass cannot be significantly enhanced. In general, lipids especially triglycerides are produced when nitrogen becomes a growth limiting factor at the late logarithmic or early stationary growth phase resulting in a low triglyceride production rate compared e.g. to yeast ethanol production. Additionally, the need of several carbons and reduced cofactors in synthesis of triacylglycerol result in low yield per used carbon. The present invention uses another route for microbial lipid production. In the present invention microbial lipid production rate and yields are enhanced, and the need of reduced cofactors from the outside of the lipid pathway is decreased. The present invention further provides lipid production that is not linked to nitrogen limitation.